The invention relates to the identification of cells and viruses in medical, industrial, or environmental samples.
Overview
Detecting, enumerating, and identifying low levels of specific cells and viruses are a cornerstone of routine medical, industrial, and environmental diagnostics. For example, samples are analyzed to detect infectious agents, cancer cells, food pathogens, microbial contaminants of pharmaceutical and cosmetic products, and microbes in water and the environment.
For simplicity, the discussion that follows focuses on routine medical diagnostics; similar methods are used for industrial and environmental applications.
Microbiological Methods
Microbial culture allows simple visual detection of microbes, (e.g., bacteria, viruses, and fungi) by exploiting their propensity to reproduce rapidly in large numbers. For example, a individual bacterial cell, which is much too small to see by eye (about one millionth of a meter), when placed in nutrient broth, can cause the broth to become visibly cloudy in less than 24 hours.
A related microbial culture technique, called microbial enumeration or colony counting, quantifies the number of microbial cells in a sample. The microbial enumeration method, which is based on in situ microbial replication, generally yields one visually detectable “colony” for each microbial cell in the sample. Thus, counting the visible colonies allows microbiologists to determine the number of microbial cells in a sample accurately. To perform microbial enumeration, bacterial cells can be dispersed on the surface of nutrient agar in petri dishes (“agar plates”) and incubated under conditions that permit in situ bacterial replication. The individual, visually undetectable, microbe replicates repeatedly to create a large number of identical daughter microbes at the physical site where the progenitor microbial cell was deposited. The daughter cells remain co-localized (essentially contiguous) with the original cell, so that the cohort of daughter cells (which may grow to tens or hundreds of millions of cells) eventually form a visible colony on the plate.
Microbial culture is a remarkably successful method, as evidenced by the fact that even after more than a century the method still dominates medical microbiology and quality control testing in industrial microbiology (e.g., pharmaceutical, food, and beverage manufacturing). The method is inexpensive, relatively simple, and ultra-sensitive. The sensitivity of microbial culture can be seen in the common test for foodborne pathogens in ground beef. A individual microscopic bacterial pathogen cell can be detected in 25 grams of ground beef using microbial culture. Another advantage of microbial culture is its ability to detect a large range of microbes of medical and industrial significance.
Some viruses can be grown in culture. Viral culture has been especially useful for fast growing bacteriophage (viruses that infect bacteria) in research applications. Viral culture is sometimes used to diagnose clinical infections although methods such as nucleic acid amplification are increasingly used instead.
Traditional microbial culture is slow—it takes time to generate the number of cells or viruses required for visual detection. The long growth period required for microbial culture is a significant problem in both healthcare and industry. For example, because it requires days to culture and identify the microbe causing a patient's blood infection, a patient with a fungal blood infection could die before anti-fungal therapy is even begun. Some infectious agents, such as the bacterium that causes tuberculosis, generally require weeks to grow in culture. The long time required for detecting M. tuberculosis can result in a patient with tuberculosis infecting many others with the highly contagious disease or the costly quarantine of patients who do not have tuberculosis. In the manufacture of food, long testing cycles can increase food spoilage. Slow microbial culture also adversely impacts the production of biopharmaceuticals and vaccines.
A number of microbial culture methods for more rapid microbial enumeration have been developed. One rapid method deposits bacterial cells on microscope slides coated with nutrient medium. Using microscopic examination, microbial growth can be detected much earlier than with the naked eye, since microscopes can detect microcolonies resulting from a small number of cell divisions. A commercialized system, the Colifast Rapid Microcolony Counter (Colifast), can detect small fluorescently labeled colonies of coliform bacteria hours before they can be seen by eye. The Colifast system achieves enhanced detection by using a fluorogenic compound (a substance that is not fluorescent until metabolized by coliform bacteria) is included in the nutrient agar media. A system for rapid enumeration of microbial colonies using bioluminescent labeling has recently been commercialized. The MicroStar system (Millipore) uses the cellular ATP in microcolonies to generate light via the action of applied luciferase enzyme and substrates. The method reduces time to detection substantially. The MicroStar imaging system has also been used in conjunction with labeled probes to identify specific bacteria (Stender, H., et al. (2001). J Microbiol Methods 46: 69-75).
Immunological Methods
Immunological tests, or immunoassays, are frequently used to identify specific cells and viruses in medical diagnostics. Immunoassays can detect the specific binding of antibodies to sites on the molecular components of targets and viruses. Serological tests are immunological assays that, rather than testing directly for antigens, test for a host immunological response to previous exposure to the antigen—for example they can test for the presence of host antibodies to particular cells and viruses. Numerous immunoassay systems are available for ranging from large automated central lab systems to over-the-counter pregnancy tests. The tests cover a broad range of formats including agglutination assays, precipitin assays, enzyme-linked immunoassays, direct fluorescence assays, immuno-histological tests, complement-fixation assays, serological tests, immuno-electrophoretic assays, and rapid “strip” tests (e.g., lateral flow and flow through tests). Immunological tests can be extremely simple and rapid. Thus, many of the most desirable tests, those that can be conducted in a physician's office or at home by the patient, are immunological tests.
Genetic Methods
Genetic methods are general and powerful tools for detecting and identifying nucleic acid molecules from cells and viruses. Revolutionary new methods for ultra-sensitively detecting and distinguishing the nucleic acid content of cells and viruses based on nucleic acid amplification have recently been developed. For example, commercial tests can detect the nucleic acids from a small number of sub-microscopic HIV virus particles (e.g., 50 particles/ml). Amplification technologies include the polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), Transcription Mediated Amplification (TMA), and rolling circle amplification (RCA). Amplification methods can deliver impressive analytical sensitivity and can be moderately rapid, for example, the Smart Cycler (Cepheid) can deliver results in an hour (Belanger, S. D., et al. (2002). Journal of Clinical Microbiology 40: 1436-40).
Microscopic Imaging Methods
Microscopic imaging is one of the most common methods for detecting cells and viruses. Targets can be visualized microscopically by labeling with stains, antibodies, or nucleic acid probes, for example. The sensitivity of microscopic analysis can be enhance using methods such as catalyzed reporter deposition (CARD), which has been used to detect single copy level viral genomes in human cells (Huang, C. C., et al., Modern Pathology 11: 971-7, 1998). Other methods for increasing the sensitivity of in situ hybridization rely on in situ amplification or replication, or signal amplification using, for example, branched DNA or dendrimer technology (e.g., Orentas, R. J., et al., Journal of Virological Methods 77: 153-63, 1999).
Multiple fluorescent labels can be used to detect several pathogens at once using in situ analysis. For example, antibodies to different category-specific antigens can be labeled with different fluorescent tags. Combinatorial labeling strategies have been also used in conjunction with microscopy to identify several bacterial pathogens simultaneously (Amann, R., et al., Journal Of Bacteriology 178: 3496-500, 1996; U.S. Pat. No. 6,007,994).
Several commercial systems have been developed that achieve high throughput in situ microscopic analysis of microscopic targets (e.g., WO 98/22618 and WO 93/21511). Typically, microbes or cells are deposited on a slide or on the bottom of the wells of a multiwell plate. Labeled targets in the wells are then imaged microscopically.
Non-Microscopic Imaging Methods
Non-microscopic imaging allows larger areas to be surveyed for the presence of cells. For example, researchers at Hamamatsu Corporation (Masuko, M., et al., FEMS Microbiol Lett 67: 231-8, 1991; Masuko, M., et al., FEMS Microbiol Lett 65: 287-90, 1991; Yasui, T., et al., Appl Environ Microbiol 63: 4528-33, 1997) developed a system that can image individual bacterial cells without magnification. The system uses an ultrasensitive photon-counting CCD camera coupled to a fiber optic system, image intensifier, and image-processor. The Elisa spot test method is a specialized technique for enumerating single cells that produce a particular antibody (or other abundantly secreted product; Logtenberg, T., et al., Immunol Lett 9: 343-7, 1985). The method's sensitivity derives from the fact that large numbers of targets (the secreted protein molecules) are localized around the secreting cell.
Flow Cytometric Methods
Flow cytometry is an important tool for characterizing cells in clinical diagnostic laboratories. Flow cytometric methods are used for quantitatively detecting particular cell types on the basis of their physical properties and their ability to bind labeled probes (e.g., stains, antibodies, or nucleic acids). Individual cells or particles are forced to flow through a narrow channel, one at a time, past a laser beam. Fluorescence emission and size/shape information is gathered by analyzing the spectrum and light scattering caused by the organism/particle. Thousands of individual cells or particles can be analyzed per minute. For example, flow cytometry is used to quantify the population sizes of classes of lymphocytes in patients with AIDS. A highly multiplexed flow cytometric method that can detect and identify non-cellular molecules such as proteins or nucleic acids has been commercialized by Luminex (U.S. U.S. Pat. No. 5,981,180).
Laser Scanning Methods
Laser scanning is a powerful and sensitive method for detecting cells and viruses deposited on surfaces. Typically, a microscopic laser beam (e.g., 20 μm in diameter) is traced over a two dimensional sample containing fluorescently labeled targets on a solid surface in a large number of successive linear passes, each slightly offset from the previous, so that the entire sample area is eventually scanned. When a fluorescently labeled target falls under the beam, a flash of emitted fluorescence is detected by a detection device such as a photomultiplier. The number and position of the microscopic targets can be obtained by laser microbeam scanning.
A number of systems for laser scanning have been developed. The ScanRDI® system (Chemunex) uses a non-specific fluorescent dye to label microbial cells on a filter (U.S. Pat. No. 5,663,057; Mignon-Godefroy, K., et al., Cytometry 27: 336-44, 1997). The fluorimetric microvolume assay technology (FMAT; PE Biosystems and Biometric Imaging; Miraglia, S., et al., J Biomol Screen 4: 193-204, 1999) has also been used to detect cells in microtiter wells. A sophisticated laser scanning system has been developed and commercialized by CompuCyte Corporation (Kamentsky, L., 2001, Laser Scanning Cytometry. In Cytometry, Z. Darzynkiewicz, H. Crissman and J. Robinsnon, eds. Methods in Cell Biology Vol. 63, Part A, 3rd ed, Series Eds. L. Wilson and P. Matsudaira. (San Diego: Academic Press)). A microbeam laser scanning system that detects individual microscopic targets in a liquid sample (e.g., whole blood) has been developed by Immunicon Corporation and collaborators at Twente University (Tibbe, A. G., et al., Nat Biotechnol 17: 1210-3, 1999).
Biochemical, Chemical, and Physical Methods
Other technologies for sensitive detection of the cells and viruses, or their molecular components, molecular components of cells and viruses include flow cytometry, mass spectroscopy, biosensors, absorbance spectroscopy, fluorescence polarization, fluorescence fluctuation spectroscopy, electrophoresis, chromatography, among many others.
Biosensor technologies also hold promise for sensitive detection of cells and viruses. Biosensors use physical methods to convert a biological event, for example binding of an antibody to an antigen, to a detectable signal. One popular biosensor used for molecular detection uses surface plasmon resonance (Mullett, W. M., et al. (2000). Methods 22: 77-91). Thermo BioStar's optical immunoassay (Schultze, D., et al. (2001). Eur J Clin Microbiol Infect Dis 20: 280-3) uses the principle of optical interference to detect binding of antigens to antibodies. The BARC biosensor technology uses magnetoresistive detection (as used for hard disk storage) of analytes tagged with single magnetic microparticles (Edelstein, R. L., et al., Biosens Bioelectron 14: 805-13, 2000).
Unmet Needs for Detection of Cells and Viruses
Although numerous disparate and powerful methods for routine detection of low levels of cells and viruses have been commercialized there are still gaps in the testing repertoire. In particular, there is a need for tests to detect very low levels of cells and viruses that are rapid, do not require laboratory growth, are user-friendly, and are cost-effective.